Biopolymer compositions, scaffolds and devices

ABSTRACT

Compositions and blends of biopolymers and copolymers are described, along with their use to prepare biocompatible scaffolds and surgically implantable devices for use in supporting and facilitating the repair of soft tissue injuries.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation Application of PCT InternationalApplication No. PCT/US2018/000119, filed May 15, 2018; which is relatedto U.S. Provisional Patent Application 62/603,026, filed May 16, 2017,the content of which is hereby incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH

The data presented in this application was supported at least in part byDARPA Contract HR0011-15-9-0006. The US government has certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates to compositions of biopolymers, such as collagen,and biodegradable co-polymers, processes for their incorporation intofibers and various scaffolds, and to implantable biocompatible devicesprepared with such compositions. More particularly, the inventionrelates to the production of biocompatible implants and devices usefulto support and facilitate the repair of soft tissue injuries, such astorn Achilles', patellar and rotator cuff tendons.

BACKGROUND OF THE INVENTION

Various approaches have been taken to develop components for implantabledevices useful as scaffolds to facilitate repair of, or to replace,damaged soft tissues such as tendons and ligaments. Such products mustfunction in a variety of challenging biomechanical environments in whichmultiple functional parameters must be addressed, among them, forexample, are compatibility, strength, flexibility and biodegradability.

Surgical repairs number around 800,000 annually in the US alone forligaments and tendons of the foot and ankle (for example, Achillestendon), shoulder (for example, rotator cuff), and knee (for example,anterior cruciate ligament), yet the current standards of care involvingthe implantation of replacement and supporting elements are generallyconsidered by medical practitioners to be less than optimal.

Leading ligament and tendon repair graft products intended to providebiocompatible soft tissue support scaffolds often involve two decadesold technologies that in some instance rely on cadaveric tissue orinvasive autografting. Allografts are supply-limited, promote scarformation, may provoke an immune response, and have poorly definedturnover rates, all of which inhibit healing. Autografting also extendssurgery time and associated trauma, and often adds a second costlyprocedure to recover the autologous tissue.

For example, the GRAFTJACKET® Regenerative Tissue Matrix is a sheet-likeproduct formed from donated allograft human dermis, asepticallyprocessed to remove cells and then freeze-dried,http://www.wright.com/footandankleproducts/graftjacket. ArthroFLEX®Decellularized Dermal Allograft is a similar acellular dermalextracellular matrix, https://www.arthrex.com/orthobiologics/arthroflex.

Among these approaches and products are those disclosed by Ratcliffe etal., U.S. Pat. No. 9,597,430 (2017), entitled “Synthetic structure forsoft tissue repair”. This patent describes various synthetic fibrillarstructures, such as a woven mesh and single or multilayer planarfibrillar forms. According to Ratcliffe, these structures can be madefrom any biocompatible polymer material capable of providing suitablemechanical properties, bioabsorbable or not. Collagen and lactide arementioned as suitable. Synthasome's “X-Repair” medical device appears tobe related and has been granted FDA 510(k) clearance by the US Food andDrug Administration (FDA), (http://www.synthasome.com/xRepair.php).

Another approach is described by Qiao et al., “Compositional and inVitro Evaluation of Nonwoven Type I Collagen/Poly-dl-lactic AcidScaffolds for Bone Regeneration,” Journal of Functional Biomaterials2015, 6, 667-686; doi:10.3390/jfb6030667. This article describeselectrospun blends of Poly-d,l-lactic acid (PDLLA) with type I collagen.Various blends are described with ratios of 40/60, 60/40 and 80/20polymer:collagen blend by weight. Qiao described a co-solvent system andreported that chemical cross linking was essential to ensure long termstability of this material in cell culture. According to Qiao, scaffoldsof PDLLA/collagen at a 60:40 weight ratio provided the greateststability over a five-week culture period.

The use of constructs for muscle implants is described by Lee et al.,U.S. Pat. No. 9,421,305 (2016), “Aligned Scaffolding System for SkeletalMuscle Regeneration.” The patent discusses an anisotropic muscle implantmade of electrospun fibers oriented along a longitudinal axis and crosslinked to form a scaffold. Cells are seeded on the fibers to formmyotubes. The fibers may be formed from natural polymers and/orsynthetic polymers. Natural polymers include, for example, collagen,elastin, proteoglycans and hyaluronan. Synthetic polymers include, forexample, polycaprolactone (PCL), poly(d,l-lactide-co-glycolide) (PLGA),polylactide (PLA), and poly(lactide-co-captrolactone) (PLCL). The fibersalso may include hydrogels, microparticles, liposomes or vesicles. Whenblended, the ratio of natural polymer to synthetic polymer is between2:1 and 1:2 by weight.

Electrospun scaffolds for generation of soft tissue are described bySensini et al., “Biofabrication of bundles of poly(lactic acid)-collagenblends mimicking the fascicles of the human Achilles tendon,”Biofabrication 9 (2017) 015025. Two different blends of PLLA andcollagen were compared with bundles of pure collagen.

SUMMARY OF THE INVENTION

The present invention relates to compositions of biopolymers andcopolymers that are biocompatible, bioactive, biodegradable andresorbable and to scaffolds and implantable devices made of suchcompositions and their blends. Such compositions and devices are usefulin supporting and facilitating the repair of soft tissue injuries.

A preferred embodiment of such a blend comprises about 10 to 50%biopolymer by weight, preferably about 15 to 40% biopolymer, morepreferably about 20 to 35% biopolymer, more preferably about 27.5 to32.5% biopolymer and most preferably about 30% biopolymer. A copolymerthat is also biocompatible, bioactive, biodegradable and resorbable ispresent in a range of about 50 to 90% by weight.

Preferred types of biopolymers include collagen, extracellular matrixproteins, fibrin, fibrinogen, gelatin and laminin, and combinationsthereof. Preferred types of collagen include native, processed,placental and recombinant forms of human, bovine, porcine and marinetelocollagen, atelocollagen and mixtures of these types of collagen. Apreferred collagen is of bovine origin. Another preferred collagen isType 1 collagen. Generally, human collagen is preferred, such as fromplacental tissue or recombinant human collagen, and mixtures thereof.The use of both telocollagen and atelocollagen are contemplated. Sourcesof marine collagen include jellyfish, sea cucumber and cuttlefish.

In one embodiment of the invention the composition comprises about 10 to50% collagen by weight, preferably about 15 to 40% collagen, morepreferably about 20 to 35% collagen, more preferably about 25 to 35%collagen, more preferably about 27.5 to 32.5% collagen and mostpreferably about 30% collagen; and a biodegradable copolymer in anamount of about 50 to 90% by weight.

A variety of copolymers are appropriate for the scaffolds and otherproducts and methods described in this specification. Preferredcopolymers are biodegradable or resorbable, such as PLLA, PDLA and PDLLAand mixtures thereof. Preferred copolymers are PDLA, low molecularweight PDLLA, mid-molecular weight PDLLA, high molecular weight PDLLAand combinations thereof.

In the compositions of the invention, the biopolymer, for example,collagen and copolymer blends may be formed into fibers. Optionally, thecompositions, fibers and other forms of implantable scaffolds may betreated with a chemical cross-linking reagent or not so treated.

In certain embodiments of the invention, particularly with techniquessuch as electrospinning, fibers range in diameter from about 150 to4,500 nm, preferably about 400 nm to 2,000 nm, more preferably about 600nm to 1,500 nm and most preferably about 750 nm to 1,200 nm. In otherembodiments, particularly such as melt electrospinning orelectrowriting, the average diameter of the fibers is in the range ofabout from about 1-200 μm, preferably about 10-100 μm, more preferablyabout 15-50 μm and most preferably about 20 μm.

The invention also relates to fibers prepared as described in thespecification and processed in the form of single or multilayersheet-like scaffolds. In one embodiment, this scaffold is composed ofaround 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more layers of substantiallyaligned telocollagen and PDLLA fibers that are each around 0.2 mm thick,with a small section of fibers laying in the transverse plane around theedges to support biaxial strength for suture retention. In anotherembodiment, this multilayer scaffold is around 4 cm×7 cm×1 mm in size.An alternative embodiment is a single layer scaffold of approximatelysimilar dimensions.

In other aspects of the invention, the compositions according to thepresent invention may be produced and used to produce scaffolds in theform films, aerosols, droplets, adhesives or porous structures.

Contemplated techniques for producing fibers and various scaffoldsinclude electrospinning, melt electrospinning, electrowriting,extrusion, spraying and 3-D printing.

Yet another aspect of the invention relates to an implantable medicaldevice for supporting the repair of a soft tissue injury in a mammalcomprising the composition of claim 1. And in other aspects, theinvention relates to methods for facilitating the repair and healing ofsoft tissue injuries through the surgical implantation of the scaffoldsand medical devices described in this specification. These methods,scaffolds and disclosed medical devices are intended for use inmammalian subjects, particularly humans. In one embodiment, theinvention relates to a method of repairing the torn Achilles tendon in ahuman subject, by surgical implanting and fastening a device such thatthe device spans and provides mechanical support to the repaired area ofthe tendon.

BRIEF DESCRIPTION OF THE TABLES

Table 1 shows a comparison of peak stress (MPa) and modulus ofelasticity (MPa) for several blends of PDLLA and collagen.

DETAILED DESCRIPTION Definitions

As used in this specification, the term:

“Biopolymer” means a naturally occurring, protein-based macromoleculenatively found in connective and other soft tissue and in theextracellular matrix, such as collagen, fibrin, fibrinogen, gelatin andlaminin.

“Co-polymer” means a synthetic polymer capable of being dissolved in abenign solvent system and mixed or blended with a biopolymer to addvarious desirable properties, for example, strength or rigidity as wouldotherwise be provided by the biopolymer alone.

“High molecular weight PDLLA” means a PDLLA product having an averageinherent viscosity (IV) of about 0.55 dL/g-4.5 dL/g or higher.

“Scaffold” means a construct formed from biopolymers and copolymers.Such constructs are preferably substantially aligned fibers formed intolayers, mats, sheets and tubes.

“Substantially aligned fibers” means that at least about half of thefibers lying within 15 to 20 degrees of a reference in a scaffold areoriented along a common axis. This is to be interpreted in contrast torandomly oriented fibers.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide synthetic fibers andrelated sheet-like and bundled fiber products for tissue engineering,particularly as soft tissue supports useful in the repair of damagedtendons and ligaments. For example, according to the present invention,a tissue-engineered ligament and tendon scaffold formed of collagen anda biodegradable polymer may be used for repair of a damaged Achillestendon. It is a further object of the invention to provide syntheticmaterials having a tensile strength, flexibility, modulus of elasticityand other biomechanical characteristics supportive of native humantendons and ligaments of similar size by way of producing scaffolds withappropriate fiber orientations suited to the particular tissue ordefect, such as a partially torn or fully torn Achilles tendon. Thisinvention provides sheet-like and bundled fiber scaffold products that,upon incorporation with host cells to form new tendon-like connectivetissue over time, possess tensile strength and modulus that willreinforce a union, such as a rejoined tendon with its torn ends suturedtogether, while not yielding or failing prior to tissue failure.

Biopolymers:

The biopolymers according to the present invention are biologicalmolecules, preferably proteins from native biological structure andextracellular matrix, that are capable of forming stable extractedproducts, particularly in the form of scaffolds prepared from biopolymerfibers. These include, by way of example, collagen, elastin, fibrin,fibrinogen and gelatin. Other proteins known to persons skilled in theart may be utilized in the methods of the present invention.

Collagen:

A preferred biopolymer is collagen. Type I Collagen used forbiocompatible scaffolds according to the present invention, as well asfor current clinical products, generally are extracted from mammaliantissues, particularly bovine and porcine tendons, although recombinantcollagen also may be used. Human placenta also is sometimes used forsuch purposes. Type I collagen has been utilized and commercialized inboth research and clinical grade products in two common forms. The morecommon collagen variants, produced with acid and enzymatic digestion ofa tissue with pepsin, are a form of collagen referred to as“atelocollagen,” as the product lacks the end-terminal regions of thecollagen protein (terminal peptide sequence of “DEKSTGISVP vs.pQLSYGYDEKSTGISVP), whereby the telopeptides are cleaved to aid inrecovery of collagen from the parent tissue. Less commonly, collagen issolubilized in mild acid to collect the collagen in solution,maintaining the telopeptides in the monomers of collagen, known as“telocollagen.”

Telocollagen has been reported to form a stronger hydrogel relative togels made of atelocollagen, although their relative strengths whengenerated as tissue engineered electrospun nanofibers have not been wellexplored. An experiment was conducted in which telocollagen andatelocollagen were dissolved using 40% acetic acid and electrospun toprepare scaffolds.

The present inventors found that strength of such scaffolds generallywas like that of native collagen, however, other properties of thescaffolds were not optimal as shown in Table 1. Accordingly, blends ofcollagen and various polymers were evaluated and experimental resultsare described below. Choosing a higher molecular weight PDLLA led to anincrease in the peak stress and modulus of elasticity of the constructs.Accordingly, the high molecular weight (HMW) PDLLA is preferred.

TABLE 1 Summary of Tensile Testing Results (as statistical mean ± SD).PDLLA MW: 75,000-120,000 PDLLA HMW: ~450,000 Bovine Tail 75% PDLLA:25%75% PDLLA:25% 80% PDLLA:20% Ligaments Telocollagen AtelocollagenTelocollagen Peak Stress (MPa) 5.6 ± 2.2  5.2 ± 0.5  3.1 ± 0.1 13.1 ±2.1  Modulus of 6.1 ± 3.1 21.19 ± 3.9 19.5 ± 4.7 65.6 ± 17.4 Elasticity(MPa)

Acid-soluble (telocollagen) and pepsin-soluble (atelocollagen) freezedried collagen are appropriate starting materials. A preferredGMP-grade, type I collagen from bovine corium is available in its nativeform from Collagen Solutions,http://www.collagensolutions.com/products/medical-grade-collagen. TheCollagen Solutions' website provides general information on the use andpreparation of collagen:http://collagensolutions.com/resource-library#technical-services.Collagen is also available from other suppliers and from variousspecies, for example, Sigma-Aldrich,http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/structural-proteins/collagen.html.

Copolymers:

A wide variety of biodegradable and bioactive copolymers have beenconsidered for use in soft tissue repair, alone or in blends with otherpolymers, and sometimes including components of native tissue such as,for example, collagen, fibrin and elastin. Of these, the presentinventors have discovered surprising biomechanical and biodegradabilityresults from the blended combination of collagen with polylactic acid,including both its L- and D-isoforms, and particularly so with itsamorphous mixture referred to as poly-DL-lactide or PDLLA.

Other copolymers that may be useful for a particular product or device,or when added to a blend of polylactide and collagen, include1,3-propanediol (PDO), polycaprolactone (PCL), poly(lactic-co-glycolicacid) (PLGA). Other useful polymers and copolymers would be known topersons skilled in the art, for example, poly(glycolic acid),polyesters, trimethylene carbonate, polydioxanone, caprolactone,alkylene oxides, ortho esters, hyaluronic acids, alginates, syntheticpolymers from natural fats and oils, and combinations thereof.

With respect to the polylactides, the PLLA isoform alone is relativelystrong but brittle rather than elastic. It persists in vivo for about 36to 48 months. A preferred PLLA is available from Sigma Aldrich.http://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/material-matters/pdf/resomer-biodegradeable-polymers.pdf.

The PDLA isoform is more elastic and not as brittle, and typically lastsfor 12 to 18 months in vivo. A preferred PDLA is available from SigmaAldrich.http://www.sigmaaldrich.com/catalog/product/SIGMA/67122?lang=en&region=US.

PDLLA lies between PLLA and PDLA in terms of strength and stability andin terms of lifespan in vivo, is in the range of about 18 to 36 months,which is long enough to be resorbed and short enough to avoidencapsulation. PDLLA is an amorphous polymer formed via polymerizationof a racemic mixture of L- and D-lactides. The precise composition ofthe polymer determines its mechanical properties and hydrolysischaracteristics.

PDLLA generally displays more favorable degradation properties, due tothe level of access of water in the amorphous material and thehydrolytic cleavage of polymer ester bonds. The present inventors havefound that PDLLA is surprisingly effective for producing fibers andimplantable support devices when blended with Type 1 collagen for theuses described in this specification.

Preferred sources of PDLLA are Polysciences, Evonik and Sigma-AldrichCo. LLC. For example, PDLLA having an inherent viscosity of 1.6-2.4 dL/gis available from Polysciences,http://www.polysciences.com/default/polyd)-lactic-acid-iv-20-28dlg,having an average molecular weight range of about 300,000 to 600,000Daltons. A lower inherent viscosity PDLLA (IV of 1.3-1.7 dL/g) isavailable from Evonik,http://healthcare.evonik.com/product/health-care/en/products/biomaterials/resorner/pages/medical-devices.aspx.A PDLLA with even lower inherent viscosity of 0.55-0.75 dL/g isavailable from Sigma-Aldrich,http://www.sigmaaldrich.com/catalog/product/sigma/p1691?lang=en&region=US,having molecular weight range of about 75,000 to 125,000 Daltons.Another preferred PDLLA with a GMP level of purity available fromCorbion (“PURASORB PDL 45”) has a relatively high inherent viscosity of4.5 dL/g,http://www.crobion.com/static/downloads/datasheets/31d/PURASORB%20PDL%2045/pdf.

Functionalization of Copolymers:

Copolymers may be pretreated with one or more functionalization reagentsto prepare the copolymer for cross-linking after extraction of thebiopolymer-co-polymer mixture by a production technique such aselectrospinning. For example, PDLLA can be functionalized throughaminolysis to add amino groups. See, for example, Min et al.,“Functionalized Poly(D,L-lactide) for Pulmonary Epithelial CellCulture,” Advanced Engineering Materials 12(4):B101-B112 (2010) athttp://onlinelibrary.wiley.com/doi/10.1002/adem.200980031/abstract.Alternatively, PDLLA can be functionalized by plasma treatment tointroduce carboxylic and amino groups in the matrix.

As a general approach, by way of example, PDLLA can be functionalizedwith OH groups prior to electrospinning. PDLLA pellets are soaked in asolution mixture of 10 mM-1M sodium hydroxide dissolved in 10-20%ethanol in milliQ water. The pellets will soak for 10-60 minutes ateither room temperature or 37 C. Following incubation, the pellets willbe rinsed in milliQ (ultrapurified) water and air dried in a biosafetyhood. The functionalized PDLLA chips could then be dissolved in anappropriate electrospinning solution as described in this specification.

Collagen-Copolymer Blends:

In preferred embodiments of the present invention, the tensile strengthof scaffolds generated from collagen, for example, telocollagen, and apolymer, for example, PDLLA, are comparable or exceed in biomechanicalproperties, for example, to that of bovine tail ligaments. However,similar blends made with atelocollagen of different sources maydemonstrate a lower tensile strength in some instances, as will beapparent to persons skilled in the art. For example, one batch ofelectrospun telocollagen with a lactide polymer was nearly 50% strongerthan atelocollagen prepared in the same way, that is about 5.5 MPa vs.about 4 MPa. Additionally, using PDLLA of a relatively higher molecularweight (450,000 vs. 75,000-120,000) more than doubled the strength ofthe construct to about 13.1 MPa.

Both telocollagen and atelocollagen blended with PDLLA were assessed forlong-term stability in tissue culture media to ensure suitability forlong-term cell culture assays. The collagen-PDLLA scaffolds showacceptable stability in culture media over 28 days of incubation. Likethe dry testing results of tensile strength tests, telocollagen blendedwith PDLLA was also surprisingly stronger mechanically (wet tested)compared to atelocollagen blends with PDLLA.

According to the present invention, a preferred composition comprisesabout 10 to 50% collagen, preferably about 15 to 40% collagen, morepreferably about 20 to 35% collagen, more preferably about 25 to 35%collagen, more preferably about 27.5 to 32.5% collagen and mostpreferably about 30% collagen by weight; with about 50 to 90% by weightof a lactide copolymer.

Type I collagen of bovine origin is preferred as a biopolymer and alactide polymer, particularly high molecular weight PDLLA, is preferredas a copolymer. Telocollagen is preferred over atelocollagen. Suchcompositions exhibit desired biomechanical performance and biostabilityparameters, such as its wettability properties.

Preparation and Processing of Collagen-Polymer Blends:

The preparation of collagen and lactide polymer blends is described withparticularity in the Examples that follow. Generally, both componentsare dissolved in hexafluoro-2-propanol (HFP). Preferably, nocross-linking reagents are added to the reagent blend prior to itsprocessing into fibers. Optionally, various conventional cross-linkingcompounds may be blended with the collagen and polymer, or the resultingmaterials may be cross-linked after electrospinning.

Electrospinning is a preferred processing technique to produce fibersfrom the inventive compositions, although other approaches will be knownto persons skilled in the art. although other approaches to separatingthe blend from the solvent system will be known to persons skilled inthe art, for example, pneumatospinning, extrusion, cold drawing orcasting. Electrospinning is a fiber production technology that drawscharged threads of polymer solutions or polymer melts into fibers ofvarious diameters and lengths. Electrospinning of collagen has beenwidely described as a one-step process for the formation of fibrousmaterials that mimic native tissue structure. Electrospinning equipmentis conventional and readily available from product brands such asNanospinner, Elmarco and SprayBase. Electrospinning sharescharacteristics of both electrospraying, conventional solution dryspinning, extrusion, or pulltrusion of fibers.

Characteristics of Fibers Made of the Inventive Compositions:

Polymer blends of preferred embodiments as described above and in theExamples below were used to product electrospun fibers. Preferred fiberdiameters are in the range from about 150-4,500 nm, preferably about 400nm to 2,000 nm, more preferably about 600 nm to 1,500 nm and mostpreferably about 750 nm to 1,200 nm. A preferred range of strength forthe fibers is about 4 to 16 MPa. The preferred Modulus of Elasticitypreferably is substantially like that of human tendons, particularly theAchilles Tendon, which is about 35-750 MPa. Within that range, about35-200 MPa for the fibers is preferred. Also, a strain to failure of50-200% (0.5 to 2.0 mm/mm) as tensile tested at 1 mm/s in hydratedcondition is preferred.

Preparation of Scaffolds:

A preferred biopolymer structure is a scaffold that is appropriate forimplantation as a support to help repair a soft tissue injury or as areplacement for such tissue, for example, a tendon or ligament.Scaffolds appropriate for implantation may be made by varioustechniques. For example, scaffolds in the form of sheets may be producedby electrospinning collagen and copolymer blends onto a high-speed drum(surface speed of around 1 to 20 m/s, for example at about 18 m/s).Fibrous sheets are readily peeled from the drum of an electrospinningapparatus in sheets or otherwise removed by conventional techniques.

Scaffolds can be vacuum dried after electrospinning to remove residualsolvents. For example, the sheets preferably are stored for about 1-3days under vacuum at about 30-37° C. to remove residual processingsolvents. The sheets then may be cut or oriented to generate secondaryand tertiary structures and, optionally, may be laminated throughwelding or suturing/sewing.

Such sheets may be laminated through welding or suturing or sewing. Ingeneral, the sheets of electrospun material are stacked. In general, thesheets of electrospun material are stacked. Then heat (30-100° C., forexample about 60° C.) is locally applied to join them. Additionalmaterial may be added into welds to reinforce material to aid in sutureretention. Optionally, an adhesion barrier may be included which wouldbe comprised of a pure polymer backing (facing away from tendon) toprevent extrinsic cell infiltration. The polymer layer may beelectrospun, cast, foamed, extruded, or produced by other conventionaltechniques.

With respect to scaffolds prepared from the fibers, the scaffold'swettability preferably shows stability in culture media over about 28days of incubation at 37° C. with 100% humidity in 5% CO₂. Generally,seeded cells should show robust cell attachment, preferably with morethan half the cells attaching to the scaffold, as described in theExamples. Initial retention of growth factors preferably issubstantially like that of human tendon, particularly the AchillesTendon.

Persons skilled in the art will be aware of appropriate techniques forthe fabrication, production and construction of three-dimensionalscaffolds according to the compositions and methods of the presentinvention. Such techniques are described, for example, by Bhatia et al.,“Microfabricated biopolymer scaffolds and method of making same,”Published US Patent Application US20050008675A1; Hoque et al.,“Extrusion based rapid prototyping technique: An advanced platform fortissue engineering scaffold fabrication,” Biopolymers 97: 83-93, 2012,https://doi.org/10.1002/bip.21701; Lu et al, “Techniques for fabricationand construction of three-dimensional scaffolds for tissue engineering,”Int. J Nanomedicine. 2013; 8: 337-350; Li et al, “3D-Printed Biopolymersfor Tissue Engineering Application,” International Journal of PolymerScience, Volume 2014, Article ID 829145,https://doi.org/10.1155/2014/829145; and Ma, “Scaffolds for tissuefabrication,” Materials Today Volume 7, Issue 5, May 2004, Pages 30-40.

Additional Processing of Scaffolds:

Generally, when a co-polymer is functionalized to provide amino groupsprior to dissolving in the solvent system, the biopolymer and co-polymermay be crosslinked with glyoxal or aldehyde crosslinking reagents afterits extraction into a scaffold. If the co-polymer is functionalized withcarboxyl groups, then EDC and other carbodiimides may be used forcrosslinking. Isocyanates react with both OH groups and amines.Therefore, isocyanate-based crosslinkers may be used to crosslink the OHgroups to each other within, for example, the functionalized PDLLA(linking an OH group to another OH group) to improve media stabilityand/or strength. Isocyanates also may be used to link collagen to OHgroups in functionalized PDLLA via the NH2 group (that is, amine group)from the collagen. Additionally, photo-crosslinkers can be used.

Additionally, the biopolymer can be physically post-processed such as bythermal annealing with or without mechanical drawing, or by a mixture ofannealing, drawing, and relaxation cycles. These physicalpost-processing steps can be applied to temper or otherwise alter thematerial properties of the resulting scaffold, such as by changing fiberdiameter, fiber alignment, and void fraction or porosity of theresulting scaffold.

Implantable Devices:

As described above, the present invention is directed to the productionand use of synthetic fibers and related sheet-like and bundled fiberproducts for tissue engineering, particularly as soft tissue supportsuseful in the repair of damaged tendons and ligaments. For example,according to the present invention, a tissue-engineered ligament andtendon scaffold formed of elongated fibers of collagen and abiodegradable copolymer may be used for repair of a damaged Achillestendon. Scaffolds according to the invention may be in the form of amat, tube, single layer sheet and multilayered sheet.

In a preferred embodiment, the invention relates to fibers prepared asdescribed above, and processed in the form of single or multilayersheet-like scaffolds. In one embodiment, this scaffold is composed ofaround 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more layers of alignedtelocollagen and PDLLA fiber blends that are each around 0.4 mm thick,with a small section of fibers laying in the transverse plane around theedges to support biaxial strength for suture retention. In anotherembodiment, this multilayer scaffold is around 4 cm×7 cm×1 mm in size.An alternative embodiment is a single layer scaffold of approximatelysimilar dimensions.

Generally, scaffolds according to the present invention are easy tohandle in the operating room or other acute care setting and are readilycut and shaped to fit and support a given soft tissue site. Scaffoldsmay be placed in proximity to or in contact with tissue that has tornand been repaired, for example with sutures, suture anchors or surgicalglue. The scaffold provides support and reinforcement of soft tissues,such as tendons and ligaments, including Achilles tendon, rotator cuff,patellar tendon, biceps tendon, and quadriceps tendons, The scaffoldshares some of the mechanical stress and load with the repaired tissue.

The fibrous and, optionally, sheet-like structure of the scaffold permithost cell and tissue ingrowth and also vascularization of the scaffold.Over time, the scaffold is absorbed and replaced by a patient's owntissues through a remodeling process or is otherwise dissolved, degradedand ultimately removed. Scaffolds may be packaged in sterile containerseither individually or in pairs or in larger quantities.

A. Sheets. Sheets can be prepared in a variety of standard sizes such as1×2, 2×2, 3×3, 2×4, 4×6, 6×9 cm and cut to customize size and shape.

B. Mesh. A randomly aligned material can be fabricated as a non-wovenmesh with isotropic fibers and isotropic material strength in standardsizes such as 1×2, 2×2, 3×3, 2×4, 4×6, 6×9 cm and cut to customize sizeand shape.

C. Wraps. Sheets or meshes can be used as an onlay or wrapped around atissue defect.

D. Sutures. The material may be synthesized as threads, yarns or othermonofilament and multifilament strands for use as a suture to hold,locate, support or reinforce a surgical site.

E. Internal brace. The material may be synthesized as threads, yarns orother monofilament and multifilament strands for use as a suture tobrace, support or reinforce a surgical site to prevent jointoverextension and reduce risk of reruptures.

EXAMPLES Example 1: Preparing 10% Atelocollagen—90% PDLLA andElectrospinning Fibers

In a glass 5 mL v-vial (Wheaton), 36.2 mg of freeze-dried atelocollagenand 324.5 mg Poly(d,l-lactide) (PDLLA) were dissolved in 3 mLHexafluoro-2-Propanol (HFP). Collagen was obtained from CollagenSolutions (San Jose, Calif.) and PDLLA was obtained from Polysciences,Inc. The vial was placed on a rocking platform shaker, such as from VWRuntil the reagents dissolved. The solution was then electrospun using a50 mm/2 inch drum disk with electric motor; a 5 mL glass syringe withglass luer having a diameter of 11.7 mm; a 2 in, 18 gauge all stainlesssteel needle and a 100 mm needle tip. The flow rate was 1.5 mL/hr, and+17.8 kV were applied to the needle. A 90 min spin time was utilized at21° C. and a relative humidity below the lower limit of detection of25%. The resulting fibers were scraped from the drum and placed in adesiccator.

Example 2: Preparing 30% Atelocollagen—70% PDLLA and ElectrospinningFibers

In a 5 mL v-vial, 72.3 mg atelocollagen and 168 mg PDLLA were dissolvedin 2 mL HFP, and then dissolved, generally according to Example 1. Then,the solution was electrospun using a 25 mm/1 in drum disk with electricmotor, a 2 mL glass syringe with glass luer having a diameter of 8.9 mm;a 2 inch, 18 gauge all stainless steel needle and a 100 mm needle tip.The flow rate was 1.5 mL/hr and +17.0-17.1 kV were applied to theneedle. A 60 min spin time was utilized at 22.2° C. and a relativehumidity less than 25%.

Example 3: Preparing 15% Telocollagen—85% PDLLA and ElectrospinningFibers

In a 5 mL v-vial, 36.0 mg telocollagen and 204 mg PDLLA were dissolvedin 2 mL HFP, and then dissolved, generally according to Example 1. Then,the solution was electrospun using a 25 mm/1 in drum disk with electricmotor, a 2 mL glass syringe with glass luer having a diameter of 8.9 mm;a 2 inch, 18 gauge all stainless steel needle and a 100 mm needle tip.The flow rate was 1.5 mL/hr and +17.8 were applied to the needle. A 60min spin time was utilized at 22.1° C. and a relative humidity less than25%.

Example 4: Preparing 35% Telocollagen—65% PDLLA and ElectrospinningFibers

In a 5 mL v-vial, 84.0 mg telocollagen and 156 mg PDLLA were dissolvedin 2 mL HFP, and then dissolved, generally according to Example 1. Then,the solution was electrospun using a 25 mm/1 in drum disk with electricmotor, a 2 mL glass syringe with glass luer having a diameter of 8.9 mm;a 2 inch, 18 gauge all stainless steel with nickel needle and a 100 mmneedle tip. The flow rate was 1.5 mL/hr and +18.0 were applied to theneedle. A 55 min spin time was utilized at 22.1° C. and a relativehumidity less than 25%.

Example 5: Preparing 25% Telocollagen—75% PDLLA and ElectrospinningFibers

A solution of 12% telocollagen in HFP was combined with 12% PDLLA inHFP; each were dissolved in separate vials. The collagen was prepared bydissolving 60.6 mg telocollagen powder (Collagen Solutions) in 0.5 mLHFP in a 5 mL vial. The PDLLA was prepared by dissolving 239.1 mg PDLLAin 2 mL HFP in a 5 mL vial. Both solutions were placed on a rockingshaker platform at maximum speed and tilt for 2½ hours. 250 uL of the12% (w/v) collagen solution were mixed with 750 uL of the 12% (w/v)PDLLA solution and the two were mixed on the platform shaker for 20minutes. Then, the solution was electrospun using a 25 mm/1 in drum diskwith electric motor, a 1 mL glass syringe with glass luer having adiameter of 8.9 mm; a 2 inch, 18 gauge needle and a 100 mm needle tip.The flow rate was 1.0 mL/hr and +20.0 to 20.1 KV were applied to theneedle. A 55 min spin time was utilized at 23.2° C. and a relativehumidity of 48%.

Example 6: Preparation of Collagen-Polymer Scaffolds

Five sheets that are each about 0.2 mm thick are laminated by weldingwith a soldering iron at about 100° C. or with a short pulse of heatfrom an impulse sealer. Additional fibers oriented orthogonally aresealed into the weld to provide reinforcement for suture retention.Average load to pull one suture through the weld is about 28.3 N, andthe peak stress is 4.1 MPa.

Example 7: Seeding of Human Tenocytes on a Scaffold of ElectrospunFibers

Human tenocytes (5×10⁴ cells/well) were suspended in serum free mediaand then seeded on the scaffolds prepared according to Example 6, above.After 15, 30, and 60 minutes in culture, the plates were gently shakenand the non-attached cells were removed. The number of non-attachedcells suspended in each well was counted, and the percentage of attachedcells on each scaffold disk was determined based on the total number ofcells seeded. Over 50% of the cells remained attached.

While certain exemplary embodiments have been described above in detail,it is to be understood that such embodiments are merely illustrative ofand not restrictive of the broad invention. It should be recognized thatthe teachings of the invention apply to a wide variety of compositionsand devices produced from the formulations and compositions described.Persons of skill in the art will recognize that various modificationsmay be made to the embodiments of the invention described above, withoutdeparting from its broad inventive scope. Thus, it will be understoodthat the invention is not limited to the embodiments or arrangementsdisclosed, but is rather intended to cover any changes, adaptations ormodifications which are within the scope and spirit of the invention asdefined by the appended claims.

REFERENCES

All documents identified in this specification, including the followingarticles, are incorporated by reference in their entireties.

-   Addad et al., “Isolation, characterization and biological evaluation    of jellyfish collagen for use in biomedical applications,” Mar    Drugs. 2011; 9(6):967-83. doi: 10.3390/md9060967. Epub 2011 Jun. 7.-   Cheng et al., “Isolation, Characterization and Evaluation of    Collagen from Jellyfish Rhopilema esculentum Kishinouye for Use in    Hemostatic Applications,” PLoS One. 2017 Jan. 19; 12(1):e0169731.    doi: 10.1371/journal.pone.0169731. eCollection 2017.-   Hochleitner et al., “Melt Electrowriting of Thermoplastic    Elastomers,” Macromol Rapid Commun. 2018 Apr. 14:e1800055. doi:    10.1002/marc.201800055.-   Hochleitner et al., “Melt electrowriting below the critical    translation speed to fabricate crimped elastomer scaffolds with    non-linear extension behaviour mimicking that of ligaments and    tendons,” Acta Biomater. 2018 May; 72:110-120. doi:    10.1016/j.actbio.2018.03.023. Epub 2018 Mar. 17.-   Hrynevich et al., “Dimension-Based Design of Melt Electrowritten    Scaffolds,” Small. 2018 Apr. 30:e1800232. doi:    10.1002/sm11.201800232.-   Huanga et al., “A review on polymer nanofibers by electrospinning    and their applications in nanocomposites,” Composites Science and    Technology, 63(15):2223-2253 (2003).-   Krishnamoorthi et al., “Isolation and partial characterization of    collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from    Puducherry coast,” Biochem Biophys Rep. 2017 Feb. 27; 10:39-45. doi:    10.1016/j.bbrep.2017.02.006. eCollection 2017 July-   Middleton et al., “Synthetic biodegradable polymers as orthopedic    devices,” Biomaterials 21:2334-2346 (2000).-   Rudolph et al., “Surface Modification of Biodegradable Polymers    towards Better Biocompatibility and Lower Thrombogenicity,”    http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142075.-   Shekhar et al., “Electrospun Collagen: A Tissue Engineering Scaffold    with Unique Functional Properties in a Wide Variety of Applications”    Journal of Nanomaterials 2011 Article ID 348268.-   Shoseyov et al., US 2012/0273993 entitled “Method of Generating    Collagen Fibers.”-   Siow et al., “Plasma Methods for the Generation of Chemically    Reactive Surfaces for Biomolecule Immobilization and Cell    Colonization—A Review,”    http://plasmatechsystems.com/about/pubs/Plasma%20Methods%20for%20Chemically%    20Reactive%20Surfaces%20for%20Biomolecule%20Immobilization.pdf-   Tham et al., “Surface Modification of Poly (lactic acid) (PLA) via    Alkaline Hydrolysis Degradation,”    https://www.researchgate.net/profile/Zuratul_Abdul_Hamid/publication/277306838_Sur    face_Modification_of_Poly_lactic_acid_PLA_via_Alkaline_Hydrolysis_Degradation/links/5566afd408aeab77721cbfa7/Surface-Modification-of-Poly-lactic-acid-PLA-via-Alkaline-Hydrolysis-Degradation.pdf-   Zagho et al., “Recent Trends in Electrospinning of Polymer    Nanofibers and their Applications as Templates for Metal Oxide    Nanofibers Preparation,” Chapter 1 in “Nanotechnology and    Nanomaterials” edited by Haider et al., ISBN 978-953-51-2822-9,    Print ISBN 978-953-51-2821-2, Published: Dec. 21, 2016 under CC BY    3.0 license.-   Zhang, Kuihua, et al. “Electrospun scaffolds from silk fibroin and    their cellular compatibility.” Journal of Biomedical Materials    Research Part A 93.3 (2010): 976-983.-   Zhong et al., “Isolation and characterization of collagen from the    body wall of sea cucumber Stichopus monotuberculatus,” J Food Sci.    2015 April; 80(4):C671-9. doi: 10.1111/1750-3841.12826. Epub 2015    Mar. 21.

1. An implantable scaffold for supporting the repair of a soft tissueinjury comprising at least one biopolymer sheet comprising substantiallyaligned biopolymer fibers, wherein the biopolymer fibers comprise: about10 to 50% by weight of a collagen selected from the group consisting ofatelocollagen, telocollagen, recombinant human collagen and mixturesthereof; and about 50 to 90% by weight of a copolymer selected from thegroup consisting of PLLA, PDLA, PDLLA, PLGA and mixtures thereof.
 2. Animplantable scaffold of claim 1, wherein the biopolymer fibers compriseabout 20 to 35% by weight of collagen and about 65 to 80% by weight ofcopolymer.
 3. An implantable scaffold of claim 2, wherein the biopolymerfibers comprise about 27.5 to 32.5% by weight of collagen and about 67.5to 72.5% by weight of copolymer.
 4. An implantable scaffold of claim 3,wherein the collagen is telocollagen and the copolymer is high molecularweight PDLLA.
 5. An implantable scaffold of claim 2, wherein thecollagen is telocollagen and the copolymer is high molecular weightPDLLA.
 6. An implantable scaffold of claim 1, wherein the collagen istelocollagen and the copolymer is high molecular weight PDLLA.
 7. Animplantable scaffold of claim 1, wherein the biopolymer fibers aretreated with a chemical cross-linking reagent.
 8. An implantablescaffold of claim 1, wherein the copolymer is PDLLA having an inherentviscosity of 1.6-2.4 dl/g.
 9. An implantable scaffold of claim 1,wherein the average diameter of the biopolymer fibers is in a range fromabout 150-4,500 nm.
 10. An implantable scaffold of claim 9, wherein theaverage diameter of the biopolymer fibers is in a range from about 400to 2,000 nm.
 11. An implantable scaffold of claim 10, wherein theaverage diameter of the biopolymer fibers is in a range from about 750to 1,200 nm.
 12. An implantable scaffold of claim 1, wherein thescaffold is vacuum-dried.
 13. An implantable scaffold of claim 1,wherein the biopolymer fibers are annealed biopolymer fibers,mechanically-drawn biopolymer fibers or both annealed andmechanically-drawn biopolymer fibers.
 14. An implantable scaffold ofclaim 1, wherein the scaffold comprises a single biopolymer sheet. 15.An implantable scaffold of claim 1, wherein the scaffold comprises aplurality of biopolymer sheets.
 16. An implantable scaffold of claim 1,wherein the scaffold is seeded with cells.
 17. An implantable scaffoldof claim 16, wherein the cells are tenocytes.
 18. An implantablescaffold of claim 1, wherein the scaffold after implantation into a hostpermits host cell and tissue ingrowth and vascularization of thescaffold.
 19. An implantable scaffold of claim 18, wherein afterimplantation the biopolymer fibers of the scaffold are absorbed andreplaced by a host's own tissues.
 20. An implantable scaffold of claim1, wherein the injured soft tissue is selected from the group of softtissues comprising tendons and ligaments.
 21. An implantable scaffold ofclaim 20, wherein the injured soft tissue is a tendon selected from thegroup consisting of Achilles tendon, rotator cuff tendon, patellartendon, biceps tendon, and quadriceps tendon.
 22. An implantablescaffold of claim 1, packaged in a sterile container.
 23. A method forfacilitating repair of a damaged tendon, comprising the step offastening an implantable scaffold according to claim 1 to the tendonsuch that the scaffold provides mechanical support to the area ofrepair.
 24. A method of claim 23, wherein the tendon selected from thegroup consisting of Achilles tendon, rotator cuff tendon, patellartendon, bicep tendon or quadricep tendon.
 25. A method for facilitatingrepair of a damaged tendon, comprising the step of fastening animplantable scaffold according to claim 13 to the tendon such that thescaffold provides mechanical support to the area of repair.
 26. Animplantable scaffold for supporting the repair of a soft tissue injurycomprising at least one biopolymer sheet comprising substantiallyaligned biopolymer fibers, wherein the biopolymer fibers comprise: about27.5 to 32.5% by weight of a collagen selected from the group consistingof atelocollagen, telocollagen, recombinant human collagen and mixturesthereof; and about 67.5 to 72.5% by weight of a copolymer selected fromthe group consisting of PLLA, PDLA, PDLLA, PLGA and mixtures thereof;and wherein the average diameter of the fibers is in a range from about750 to 1,200 nm.
 27. An implantable scaffold of claim 26, wherein thecollagen is telocollagen and the copolymer is high molecular weightPDLLA.
 28. An implantable scaffold of claim 26, wherein the fibers ofthe biopolymer sheet are annealed fibers, mechanically-drawn fibers orboth.
 29. A method for facilitating repair of a damaged tendon,comprising the step of fastening an implantable scaffold according toclaim 26 to the tendon such that the scaffold provides mechanicalsupport the area of tendon repair.
 30. A method of claim 29, wherein thetendon is selected from the group consisting of Achilles tendon, rotatorcuff tendon, patellar tendon, biceps tendon and quadriceps tendon.